Cloning And Functional Analysis Of CsWIN1,a Transcription Factor Regulated The Wax Synthesis In Cucumber

Plant epidermal wax forms the hydrophobic layer of the plant’s aerial organ,which is the first barrier to protect plants from mechanical damage and pathogen invasion.It is very sensitive to environmental stress,and it can resist the stress of external environment by adjusting its own structure and contents,which is of great significance to plant growth and development and adaptation of environment.In addition,the epidermal wax can also change the gloss of the plant surface.Cucumber fruit luster traits is one of its important appearance quality traits,which is of great significance to the product value of cucumber.In this study,the homologous gene of WIN1/SHINE1 was cloned from cucumber and named CsWIN1.Genetic structure and amino acid sequence analysis found that the full length of CsWIN1 is 1456 bp and contained two exons and one intron.The coding region sequence(CDS)is 741 bp,encoding 247 amino acids.CsWIN1 and Arabidopsis WIN1/SHINE1 gene sequence similarity reached 59%,which contained an AP2 conservative domain.The results of fluorescence quantitative PCR showed that CsWIN1 expressed at high levels in the leaves and young fruits of cucumber plants.The specific high expression of CsWIN1 in the leaves indicated that the function of it may be related to the development of leaf epidermal cells.At the early stage of development,the expression of CsWIN1 gradually increases with the maturation of fruit,but the relative low expression of floral organs before flowering suggested that CsWIN1 may function in the early stages of fruit development.Subcellular localization analysis suggested that the CsWIN1 protein is located in the nucleus.The CsWIN1 overexpression in the transgenic plants of Arabidopsis thaliana caused a similar phenotype to WIN1/SHINE1 overexpressing lines of Arabidopsis.The expression analysis of CsWIN1 transgenic lines indicated that the expressions of waxy related genes such as CER1,CER2 and KCS1 were regulated.These results suggested that the function of CsWIN1 is conserved in the regulation of epidermal wax synthesis through regulating the expression of downstream genes related to wax synthesis to affect wax biosynthesis and metabolism.In addition,transcriptional activation assay of dual luciferase reporter gene revealed that Tril,a key gene controlling the development of trichome and determining the early stages of epidermal cell fate,activated CsWIN1 expression by activating the expression of CsWIN1pro:LUC,indicating that the development of cucumber epidermal hairs may be related to the formation of epidermal wax.At present,there are few reports on cucumber epidermal wax,and the synthetic pathways and related molecular mechanisms of epidermal wax in cucumber are still unclear.In this study,the preliminary verification of the cloning and function of cucumber CsWIN1 laid the foundation for the further study of the important quality traits of cucumber epidermal wax and its relationship with the development of epidermal hairs.